Method of treating restenosis using bisphosphonate nanoparticles

ABSTRACT

A method of treating or preventing restenosis by administering to an individual an effective amount of an active ingredient comprising a bisphosphonate particle or a bisphosphonate particulate. The bisphosphonate may be encapsulated, embedded or adsorbed within the particle, dispersed uniformly in the polymer matrix, adsorbed on the particle surface, or in combination of any of these forms. The particles include liposomes or inert polymeric particles, such as microcapsules, nanocapsules, nanoparticles, nanospheres, or microparticles. The particulates include any suspended or dispersed form of the bisphosphonate which is not encapsulated, entrapped, or adsorbed within a polymeric particle. The particulates include suspended or dispersed colloids, aggregates, flocculates, insoluble salts and insoluble complexes of the active ingredient. The active ingredient effects restenosis by inhibiting the growth and proliferation of the cell types involved in the restenotic cascade, such as macrophages/monocytes, fibroblasts and smooth-muscle cells.

[0001] This application is a continuation-in-part of co-pendingapplication Ser. No. 09/743,705 filed on Mar. 2, 2001, which is a 35U.S.C §371 filing of PCT application no. PCT/IL99/00387 filed on Jul.14, 1999, which is a continuation-in-part of Israeli application no.125336 filed on Jul. 14, 1998.

FIELD OF THE INVENTION

[0002] The present invention is concerned with compositions capable ofpreventing, inhibiting or reducing restenosis (sometimes referred to inthe art as “accelerated arteriosclerosis” and “post-angioplastynarrowing”). Specifically, the invention relates to the use ofbisphosphonate (“BP”) nanoparticles (“NP”) to effectively treatrestenosis.

BACKGROUND OF THE INVENTION

[0003] Over the past decade, mechanical means of achievingrevascularization of obstructive atherosclerotic vessels have beengreatly improved. Percutaneous transluminal coronary angioplasty (PTCA)procedures include, but are not limited to, balloon dilatation,excisional atherectomy, endoluminal stenting, rotablation and laserablation. However, revascularization induces thrombosis, and neointimalhyperplasia, which in turn cause restenosis in a substantial proportionof coronary arteries after successful balloon angioplasty and inaortacoronary saphenous vein bypass graft and other coronary grafts.Furthermore, intimal hyperplasia causes restenosis in many superficialfemoral angioplasties, carotid endarterectomies, and femoro-distal veinbypasses. Restenosis is the formation of new blockages at the site ofthe angioplasty or stent placement or the anastomosis of the bypass. Asa result, the patient is placed at risk of a variety of complications,including heart attack or other ischemic disease, pulmonary embolism,and stroke. Thus, such procedures can entail the risk of precisely theproblems that its use was intended to ameliorate. The introduction ofendovascular stents has reduced the incidence of restenosis, but thisproblem still remains significant, since restenosis or “over exuberant”tissue healing may occur at the site of stent placement. (Waller, B. F.et al., 1997, Clin-Cardiol., 20(2):153-60; Anderson, W. D et al., 1996,Curr-Opin-Cardiol., 11(6):583-90; Moorman, D. L. et al., 1996,Aviat-Space- Environ-Med., 67(10):990-6; Laurent, S. et al., 1996,Fundam. Clin. Phamacol. 10(3):243-57; Walsh, K. et al., 1996,Semin-Interv-Cardiol., 1(3):173-9; Schwartz, R. S., 1997,Semin-Interv-Cardiol., 2(2):83-8; Allaire, E. et al., 1997, Ann. Thorac.Surg., 63:582-591; Hamon, M. et al., 1995, Eur. Heart J., 16:33s-48s;Gottsauner-Wolf, M., et al., 1996, Clin. Cardiol., 19:347-356).

[0004] Despite extensive research on the incidence, timing, mechanismsand pharmacological interventions in humans and animal models to date,no therapy exists which consistently prevents coronary restenosis(Herrman, J. P. R. et al., 1993, Drugs, 46:18-52; Leclerc, G. et al.,1995, Elsevier Science, 722-724, Topol, E., 1997, The NY Academy ofSciences, 225-277). Compositions and methods for the reduction orprevention of restenosis are still greatly desired. Accordingly, itwould be desirable to develop novel compositions and methods that areeffective in treating restenosis and preventing its reoccurrence.

[0005] Bisphosphonates (“BPs”) (formerly called diphosphonates) arecompounds characterized by two C—P bonds. If the two bonds are locatedon the same carbon atom (P—C—P) they are termed geminal bisphosphonates.The BPs are analogs of the endogenous inorganic pyrophosphate which isinvolved in the regulation of bone formation and resorption. The termbisphosphonates is generally used for geminal and non-geminalbisphosphonates. The BPs may at times form polymeric chains. BPs act onbone because of their affinity for bone mineral and also because theyare potent inhibitors of bone resorption and ectopic calcification. BPshave been clinically used mainly as (a) antiosteolytic agents inpatients with increased bone destruction, especially Paget's disease,tumor bone disease and osteoporosis; (b) skeletal markers for diagnosticpurposes (linked to ^(99m)Tc); (c) inhibitors of calcification inpatients with ectopic calcification and ossification, and (d) antitartaragents added to toothpaste (Fleisch, H., 1997, in: Bisphosphonates inbone disease. Parthenon Publishing Group Inc., 184-186). Furthermore,being highly hydrophilic and negatively charged, BPs in their free formare almost incapable of crossing cellular membranes.

SUMMARY OF THE INVENTION

[0006] In one embodiment, the present invention relates to a method oftreating or preventing restenosis by administering to an individual aneffective amount of an active ingredient comprising a bisphosphonate, abisphosphonate salt, a bisphosphonate ester, or a bisphosphonatecomplex, wherein the active ingredient is in a particle dosage form.

[0007] In a further embodiment, the present invention relates to amethod of treating or preventing restenosis by administering to anindividual an effective amount of an active ingredient comprising abisphosphonate, an insoluble bisphosphonate salt, an insolublebisphosphonate ester, or an insoluble bisphosphonate complex, whereinthe active ingredient is in a free particulate dosage form.

[0008] In a “particle” dosage form, the active ingredient may beencapsulated, entrapped, embedded, or adsorbed within the particle,dispersed in the polymer matrix, adsorbed or linked on the particlesurface, or in combination of any of these forms. The particles include,but are not limited to, inert polymeric particles, such asmicrocapsules, nanocapsules, nanospheres, microspheres, nanoparticles,microparticles, or liposomes. In a “free particulate” dosage form, theactive ingredient includes any suspended or dispersed particulate formof the active ingredient itself which is not encapsulated, entrapped oradsorbed within a polymeric particle. Free particulates include, but arenot limited to, colloids, aggregates, flocculates, insoluble salts, andinsoluble complexes. Additionally, in both the particle and freeparticulate dosage forms, suspending agents and stabilizers may be used.Effective phagocytosis of both the bisphosphonate particles and thebisphosphonate free particulates by the monocytes/macrophages can affectthe activity of such phagocytic cells. The active ingredient affectsrestenosis by inhibiting phagocytic cells involved in the restenoticcascade, such as macrophages/monocytes and fibroblasts. The deliverysystem affects smooth-muscle cells (SMC) and extracellular matrixproduction indirectly by inhibiting the cells that trigger theirmigration and/or proliferation. Nevertheless, a direct effect on SMC mayalso occur. The active ingredient may be administered by any route whicheffectively transports the active compound to the desirable site ofaction. In a preferred embodiment, the mode of administration includesintra-arterial, intravenous or subcutaneous administration.

[0009] In a further embodiment, the present invention includes a methodof treating or preventing restenosis by administering to an individual,an effective amount of any compound or composite known to inactivate orinhibit blood monocytes and tissue macrophages, thereby treating orpreventing restenosis.

[0010] In a further embodiment, the present invention includes apharmaceutical composition comprising an active ingredient selected fromthe group consisting of a bisphosphonate particle, a bisphosphonateparticulate, or a salt, ester, or complex of bisphosphonate, togetherwith a pharmaceutically acceptable carrier, stabilizer or diluent forthe prevention or treatment of vascular restenosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGS. 1-3 are bar graphs of results demonstrating the effect ofclodronate encapsulated in liposomes on the reduction of restenosis inan experimental rat carotid catheter injury model as compared to theeffect of control liposomes which did not contain clodronate on the samerats. In these figures:

[0012]FIG. 1 shows the mean neointimal area to the area of the media inrats treated with clodronate containing liposomes as compared to ratstreated with control liposomes. The medial area is the differencebetween the total arterial area and the original lumen area.

[0013]FIG. 2 shows the % stenosis in rats treated with clodronatecontaining liposomes as compared to the % stenosis in rats treated withcontrol liposomes.

[0014]FIG. 3 shows the extent of medial area as an indirect index ofsmooth muscle cell viability and determined as the difference betweenthe total arterial area and the original lumen area (External elasticlamina bound area—Internal elastic lamina bound area) in rats treatedwith clodronate containing liposomes as compared to rats treated withcontrol liposomes only.

[0015]FIG. 4 illustrates the antirestenotic effects of liposomalclodronate in the balloon-injured rat and atherosclerotic rabbit carotidarterial models.

[0016]FIG. 5 tabulates the characteristics of a typical formulation ofISA encapsulated nanoparticles.

[0017]FIG. 6 illustrates the effect of ISA, specifically, free ISA,Ca⁺²-ISA salt, and ISA encapsulated in nanoparticles, on the growth ofRAW 264 cells in vitro.

[0018]FIG. 7 illustrates the effect of 50 μM Ca⁺2-ISA salt formulations,specifically, Ca²-ISA adsorbed on blank NP, Ca⁺²-ISA salt and polyvinylalcohol (PVA), and Ca⁺²-ISA salt, on the proliferation of RAW 264 cellsin vitro.

[0019]FIGS. 8a and 8 b are bar graphs of results demonstrating theeffect of ISA encapsulated in nanoparticles on the reduction ofrestenosis in an experimental rat carotid catheter injury model ascompared to the effect of control nanoparticles (i.e., blank NP) whichdid not contain ISA, on the same rats. In these figures:

[0020]FIG. 8a illustrates the % stenosis in rats treated with ISAencapsulated nanoparticles as compared to the % stenosis in rats treatedwith control nanoparticles; and

[0021]FIG. 8b illustrates the mean neointimal area to the area of themedia ratio in rats treated with ISA encapsulated in nanoparticles ascompared to rats treated with control nanoparticles. The medial area isthe difference between the total arterial area and the original lumenarea.

[0022]FIG. 9 tabulates the characteristics of a typical formulation ofalendronate encapsulated in nanoparticles.

[0023]FIG. 10 tabulates the effect of alendronate loaded nanoparticleson the proliferation of RAW 264 cells.

[0024]FIGS. 11, 12a and 12 b are bar graphs of results demonstrating theeffect of alendronate encapsulated in nanoparticles on the reduction ofrestenosis in a hypercholesterolemic balloon-injured rabbit model ascompared to the effect of control nanoparticles which did not containalendronate on the same rats via subcutaneous administration. The graphsalso compare the effect of subcutaneous (SC) and intravenous (IV)administration in reducing restenosis. In these figures:

[0025]FIG. 11 illustrates the % stenosis in rats treated withalendronate loaded nanoparticles as compared to the % stenosis in ratstreated with control nanoparticles, wherein the particles wereadministered subcutaneously;

[0026]FIG. 12a compares the % stenosis in rats treated with: 1.5 mg/kgof alendronate loaded nanoparticles via SC administration, 0.15 mg/kg ofalendronate loaded nanoparticles via SC administration and 0.15 mg/kg ofalendronate loaded nanoparticles via IV administration; and

[0027]FIG. 12b illustrates the mean neointimal to medial area ratio(N/M) in rats treated with ISA loaded nanoparticles as compared to ratstreated with control nanoparticles, and also compares the meanneointimal area to medial area ratio (N/M) in rats treated with: 1.5mg/kg of alendronate loaded nanoparticles via SC administration, 0.15mg/kg of alendronate loaded nanoparticles via SC administration and 0.15mg/kg of alendronate loaded nanoparticles via IV administration. Themedial area is the difference between the total arterial area and theoriginal lumen area.

[0028]FIG. 13 illustrates the effect of alendronate encapsulated innanoparticles on the number of monocytes in the human blood followingincubation for 24 hours.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention relates to compositions and methods forreducing, delaying or eliminating restenosis. Reducing restenosisincludes decreasing the thickening of the inner blood vessel lining thatresults from stimulation and proliferation of smooth muscle cell andother cell migration and proliferation, and from extracellular matrixaccumulation, following various angioplasty procedures. Delayingrestenosis includes delaying the time until angiographic re-narrowing ofthe vessel appears or until the onset of clinical symptoms which areattributed to stenosis of this vessel. Eliminating restenosis followingangioplasty includes reducing hyperplasia to an extent which is lessthan 50% of the vascular lumen, with lack of clinical symptoms ofrestenosis. Methods of intervening include re-establishing a suitableblood flow through the vessel by methods such as, for example, repeatangioplasty and/or stent placement, or CABG.

[0030] The present invention includes a method of treating or preventingrestenosis by administering to an individual, an effective amount of anycompound or composite known to inactivate or inhibit blood monocytes andtissue macrophages.

[0031] One example of a group of drugs useful in the present inventionto inhibit restenosis, are bisphosphonates (“BPs”). BPs inhibit smoothmuscle cell migration and proliferation by transiently depleting and/orinactivating cells that are important triggers in the restenosiscascade, namely macrophages and/or monocytes. Bisphosphonates, whenencapsulated in liposomes or nanoparticles in a “particle” dosage form,or when in a “free particulate” dosage form, such as, for example, inaggregates of a specific size, are taken-up, by way of phagocytosis,very efficiently by the macrophages and monocytes, and to some extent byother cells with phagocytic activity such as fibroblasts. Once insidethe macrophages, the liposomal structure of the cell is disrupted andthe bisphosphonates are released, thereby inhibiting the activity and/orkilling the macrophages. Since macrophages, in their normal state, arerecruited to the areas traumatized by angioplasty or other intrusiveintervention and initiate the proliferation of smooth-muscle cells(SMC), inhibiting the macrophages' activity inhibits the migration andproliferation of SMC. Once released inside after being taken-up by themacrophages, the bisphosphonates have a sustained inhibitory activity onthe macrophages. Thus, prolonged release of the bisphosphonates is notrequired in order to sustain inhibition. Accordingly, the method ofinhibiting or reducing restenosis by administering a bisphosphonate in aparticle or free particulate form is preferably a systemic therapy, inthat the bisphosphonate particles and particulates target thecirculating monocytes and macrophages.

[0032] It should be noted, however, that some bisphosphonate particlesand particulates may have a direct effect on SMC activity. Additionally,some of the bisphosphonate particles and particulates may alsoinactivate other phagocytic cells and cells of the white-blood celllineage in the body, such as liver and spleen macrophages andmacrophages in the arterial walls.

[0033] Furthermore, the delivery system of the present invention notonly retains the BP for a sufficient time so that the free BP is notreleased in the body fluids, but also efficiently discharges the drugwithin the cell. The free BP drug, as opposed to BP particles, isineffective since it is not taken-up by phagocytic cells.

[0034] An additional example of a group of drugs useful in the presentinvention to inhibit restenosis are inactivators ofmonocytes/macrophages, such as gallium or gold.

[0035] In accordance with the present invention, a bisphosphonate or acompound or composite which inactivates monocytes/macrophages(collectively herein: “active ingredient”) is used for treatment orprevention of vascular restenosis. The term bisphosphonate as usedherein, denotes both geminal and non-geminal bisphosphonates. The term“active ingredient” encompasses in its scope, not only BP and compoundswhich inactivate monocytes/macrophage, but also polymeric chains of theBPs and the monocyte/macrophage inactivators, particularly such chainsconsisting of up to 40 BP monomers. Preferred active ingredients arecompounds of the following formula (1)

[0036] wherein

[0037] R₁ is H, OH or a halogen atom; and

[0038] R₂ is halogen; linear or branched C₁-C₁₀ alkyl or C₂-C₁₀ alkenyloptionally substituted by heteroaryl or heterocyclyl C₁-C₁₀ alkylaminoor C₃-C₈ cycloalkylamino where the amino may be a primary, secondary ortertiary; —NHY where Y is hydrogen, C₃-C₈ cycloalkyl, aryl orheteroaryl; or R₂ is —SZ where Z is chlorosubstituted phenyl orpyridinyl.

[0039] The present invention thus provides the use of said activeingredient, a complex of said active ingredient or a pharmaceuticallyacceptable salt or ester thereof for the preparation of a compositionfor the prevention or treatment of vascular restenosis. In oneembodiment, the composition comprises a “particle” dosage form, whereinthe active ingredient is encapsulated, embedded, and/or adsorbed withina particle, dispersed in the particle matrix, adsorbed or linked on theparticle surface, or in combination of any of these forms. The particleincludes any of the liposomes, microparticles, nanoparticles,nanospheres, microspheres, microcapsules, or nanocapsules known in theart (M. Donbrow in: Microencapsulation and Nanoparticles in Medicine andPharmacy, CRC Press, Boca Raton, Fla., 347). The term particle includesboth polymeric and non-polymeric preparations of the active ingredient.In a further embodiment, the composition comprises a “free particulate”dosage form of the active ingredient, such as an insoluble salt,insoluble ester, or insoluble complex of the active ingredient.Typically, “insoluble” refers to a solubility of one (1) part of acompound in more than ten-thousand (10,000) parts of a solvent. A “freeparticulate” dosage form includes any insoluble suspended or dispersedparticulate form of the active ingredient which is not encapsulated,entrapped or adsorbed within a polymeric particle. Free particulatesinclude, but are not limited to, suspended or dispersed colloids,aggregates, flocculates, insoluble salts and insoluble complexes. In yeta further embodiment, the composition comprises polymeric chains of theactive ingredient. In a preferred embodiment, the composition comprisesa bisphosphonate nanoparticle.

[0040] The present invention also provides a method of treatment ofrestenosis, comprising administering to an individual in need aneffective amount of said active ingredient, a complex thereof or apharmaceutically acceptable salt or ester thereof.

[0041] The present invention still further provides a pharmaceuticalcomposition for the prevention or treatment of restenosis comprising, aneffective amount of the active ingredient, a complex or a salt thereof,optionally together with a pharmaceutically acceptable carrier ordiluent. Carriers include, but are not limited to, liposomes, particles,and lipid particles.

[0042] The term “effective amount” denotes an amount of the activeingredient, which is effective in achieving the desired therapeuticresult, namely prevention, reduction, or elimination of vascularrestenosis. The effective amount may depend on a number of factorsincluding: weight and gender of the treated individual; the type ofmedical procedure, e.g. whether the vascular restenosis to be inhibitedis following balloon angioplasty, balloon angioplasty followed bydeployment of a stent; the mode of administration of the activeingredient (namely whether it is administered systemically or directlyto the site); the type of carrier being used (e.g. whether it is acarrier that rapidly releases the active ingredient or a carrier thatreleases it over a period of time); the therapeutic regime (e.g. whetherthe active ingredient is administered once daily, several times a day,once every few days, or in a single dose); clinical factors influencingthe rate of development of restenosis such as diabetes, smoking,hypercholesterolemia, renal diseases; anatomical factors such as whetherthere is severe preangioplasty stenosis, total occlusion, left anteriordescending coronary artery location, saphenous vein graft lesion, longlesions, multivessel or multilesion PTCA; and on the dosage form of thecomposition. Moreover, procedural variables may also have bearing on thedosage, such as greater residual stenosis following PTCA, severedissection, intimal tear, appropriate size of balloon, and the presenceof thrombus. The artisan, by routine type experimentation should have nosubstantial difficulties in determining the effective amount in eachcase.

[0043] The invention is applicable for the prevention, reduction ortreatment of vascular restenosis and mainly, but not limited to,coronary restenosis after angioplasty. Vascular restenosis primarilyresults from various angioplasty procedures including balloonangioplasty, intravascular stent deployment or other methods ofpercutaneous angioplasty (including angioplasty of coronary arteries,carotid arteries, and other vessels amenable for angioplasty) as well asfor restenosis resulting from vascular graft stenosis (e.g. followingby-pass surgery) (Braunwald, E., 1997, Heart Disease in: A textbook ofcardiovascular medicine; 5th Ed., W. B. Saunders Company: Philadelphia).

[0044] In addition, the invention is also applicable for use inprevention, reduction or treatment of vascular restenosis in peripheralarteries and veins.

[0045] One exemplary application of the invention is to prevent andtreat in-stent restenosis. It is a widely acceptable medical procedureto deploy a stent within a blood vessel within the framework of anangioplastic procedure, to support the walls of the blood vessel.However, very often restenosis occurs notwithstanding the presence ofthe stent within the blood vessel. In accordance with the invention, theabove noted active ingredient may be administered, either systemicallyor directly to the site, in order to prevent or inhibit such restenosis.The active ingredient may be formulated in a manner allowing itsincorporation onto the stent which, in fact, yields administration ofsaid active ingredient directly at the site. The active ingredient maybe formulated in that manner, for example, by including it within acoating of the stent. Examples of coatings are polymer coatings, (e.g.,made of polyurethane), gels, fibrin gels, hydrogels, carbohydrates,gelatin, or any other biocompatible gel.

[0046] The active ingredient used in accordance with the invention maybe formulated into pharmaceutical compositions by any of theconventional techniques known in the art (see for example, Alfonso, G.et al., 1995, in: The Science and Practice of Pharmacy, Mack Publishing,Easton Pa., 19th ed.). The compositions may be prepared in various formssuitable for injection, instillation or implantation in body such assuspensions of the nanoparticles, as in a coating of a medical devicesuch as a stent (see above). In addition, the pharmaceuticalcompositions of the invention may be formulated with appropriatepharmaceutical additives for parental dosage forms. The preferredadministration form in each case depends on the desired delivery mode,which is usually that which is the most physiologically compatible withthe patient's condition and with the other therapeutic treatments whichthe patient currently receives.

[0047] In a preferred embodiment of the invention, the active ingredientis selected from the group of bisphosphonates. One preferred activeingredient for this group is the compound clodronate,(dichloromethylene) diphosphonic acid, (Fleisch, H., 1997, in:Bisphosphonates in bone disease. Parthenon Publishing Group Inc.,184-186) having the following formula (II):

[0048] Clodronate was previously described for use in the treatment ofhypercalcemia resulting from malignancy in the treatment of tumorassociated osteolysis (Fleisch, H., 1997, in: Bisphosphonates in bonedisease. Parthenon Publishing Group Inc., 184-186). Clodronate was alsofound to inhibit macrophages in vitro and to suppress macrophageactivity in the spleen and liver tissues of mice. (Mönkkönen, J. et al,1994, J. Drug Target, 2:299-308; Mönkkönen, J.et al., 1993, Calcif.Tissue Int., 53:139-145).

[0049] Other preferred active ingredients of this group are etidronateand tiludronate having the following formulae (III) and (IV)respectively:

[0050] Additional BPs having activities similar to that of clodronateare also preferred in accordance with the invention. Such BPs may beselected on the basis of their ability to mimic the biological activityof clodronate. This includes, for example: in vitro activity ininhibiting phagocytic activity of phagocytic cells, e.g. macrophages andfibroblasts; inhibition of secretion of IL-1 and/or IL-6 and/or TNF-αfrom macrophages; in vivo activity, e.g. the ability of the tested BP toprevent or reduce restenosis in an experimental animal model such as,for example, the rat or rabbit carotid catheter injury model describedin Example 1 below, or porcine model of restenosis.

[0051] The most preferred group of active ingredients in accordance withthe invention are the amino-BPs and any other nitrogen-containing BPshaving the following general formula (V):

[0052] wherein X represents C₁-C₁₀ alkylamino or C₃-C₈ cycloalkylamino,where the amino may be primary, secondary or tertiary; or X representsNHY where Y is hydrogen, C₃-C₈ cycloalkyl, aryl or heteroaryl.

[0053] The BPs belonging to this group are believed not to bemetabolized and have been shown at relatively low concentrations toinduce secretion of the interleukin IL-1 and cause, at relatively highconcentrations, apoptosis in macrophages (Mönkkönen, J.et al., 1993,Calcif Tissue Int., 53:139-145). Preferred BPs belonging to this groupare for example, pamidronate and alendronate having the followingformulae (VI) and (VII), respectively.

[0054] Although the geminal BPs are preferred BPs in accordance with theinvention, non-geminal BPs, monophosphonates of BPs, termed generally asphosphonates may also be used as active ingredients in accordance withthe invention.

[0055] Additional bisphosphonates include, but are not limited to,3-(N,N-dimethylamino)-1-hydroxypropane-1,1-diphosphonic acid, e.g.dimethyl-APD; 1-hydroxy-ethylidene-1,1-bisphosphonic acid, e.g.etidronate; 1-hydroxy-3 (methylpentylamino)-propylidene-bisphosphonicacid, (ibandronic acid), e.g. ibandronate;6-amino-1-hydroxyhexane-1,1-diphosphonic acid, e.g. amino-hexyl-BP;3-(N-methyl-N-pentylamino)-1-hydroxypropane-1,1-diphosphonic acid, e.g.methyl-pentyl-APD; 1-hydroxy-2-(imidazol-1-yl)ethane-1,1-diphosphonicacid, e.g. zoledronic acid;1-hydroxy-2-(3-pyridyl)ethane-1,1-diphosphonic acid (risedronic acid),e.g. risedronate;3-[N-(2-phenylthioethyl)-N-methylamino]-1-hydroxypropane-1,1-bishosphonicacid; 1-hydroxy-3-(pyrrolidin-1-yl)propane-1,1-bisphosphonic acid,1-(N-phenylaminothiocarbonyl)methane-1,1-diphosphonic acid, e.g. FR78844 (Fujisawa); 5-benzoyl-3,4-dihydro-2H-pyrazole-3,3-diphosphonicacid tetraethyl ester, e.g. U81581 (Upjohn); and1-hydroxy-2-(imidazo[1,2-a]pyridin-3-yl)ethane-1,1-diphosphonic acid,e.g. YM 529.

[0056] Thus, suitable bisphosphonates for use in the present inventioninclude the acid compounds presented above, any acceptable saltsthereof, and crystalline and amorphous BPs. Additionally, the mostpreferred bisphosphonates are the amino-bisphosphonates such asalendronate, zolendronate, and risendronate.

[0057] The composition of the invention may comprise said activeingredient either encapsulated within a particle, adsorbed on theparticle surface, complexed with metal cations such as calcium,magnesium or organic bases, formed into non-soluble salts or complexes,or polymerized to yield polymers of up to 40 monomers. The salts may besodium, potassium, ammonium, gallium or calcium salts or salts formedwith any other suitable cation (e.g. organic amino compounds). The saltsor polymers may be in a micronized particulate form having a diameterwithin the range of about 0.01-1.0 μm, preferably within a range ofabout 0.1-0.5 μm. The active ingredients in their salt form may be withor without water of crystallization (hydrous and anhydrous).Additionally, additives such as polyvinyl alcohol (PVA), pluronics, andother surface active agents, may be used to stabilize the salt and orcomplex to establish a colloidal or nano-size suspension. In oneembodiment for example, the composition may comprise a Ca-BP salt and orcomplex.

[0058] In one embodiment of the invention, the active ingredient isencapsulated in liposomes. The liposomes may be prepared by any of themethods known in the art (regarding liposome preparation methods seeMönkkönen, J. et al, 1994, J. Drug Target, 2:299-308, and Mönkkönen,J.et al., 1993, Calcif. Tissue Int., 53:139-145). The liposomes may bepositively charged, neutral or negatively charged (negatively chargedliposomes being currently preferred), and may be single ormultilamellar. Suitable liposomes in accordance with the invention arepreferably non toxic liposomes such as, for example, those prepared fromphosphatidyl-choline phosphoglycerol, and cholesterol, e.g. as describedbelow. In many cases, use of liposomal delivery results in enhanceduptake of the active ingredient by cells not only via endocytosis butalso via other pathways such as fusion (such uptake may play a role inthe therapeutic effect). The diameter of the liposomes used may rangefrom 0.15 to 300 nm. However, this is non-limiting, but merely anexample, and liposomes of other size ranges may also be used.

[0059] In a preferred embodiment, the active ingredient orbisphosphonate may be encapsulated or embedded in inert particles. In afurther embodiment, the active ingredient may be adsorbed onto thesurface of, or adsorbed within, a blank particle, wherein a blankparticle is a particle which has no drug encapsulated or embeddedtherein. Alternatively, the active ingredient may form a particulate,which includes a colloid, aggregate, flocculate or other such structureknown in the art for the preparation of particulates of drugs.Furthermore, such particulates may be aggregates of the polymerizedactive ingredient.

[0060] Particulates of the active ingredient may be obtained by using aninsoluble salt or complex that can be obtained in-situ, i.e., startingwith the soluble drug and “salting-out” the drug by adding for example,Ca at the appropriate concentration and pH. The dispersed or freeparticulates are formed and then stabilized by the aid of surface activeagents, suspending agents, deflocculating agents or by thickeningagents, such those used in gels. The active ingredient may be furtherprecipitated by adding a trivalent cation, for example, gallium, therebyforming a precipitate of gallium-BP salt/complex.

[0061] The active ingredient may be encapsulated within or adsorbed ontoparticles, e.g., nanoparticles by utilizing, for example, a modifiednano-precipitation method. In this embodiment of the invention, thepolymeric nanoparticle containing the active ingredient is formed bymixing water and organic solutions of the drug and polymer (PLGA orother polymers), respectively. Thus, the nanoparticle containing drugformed is suspended in water and can be lyophilized. Additionally, theactive ingredient may be entrapped or adsorbed into blank polymericnanoparticles, and/or adsorbed on the surface of the blank polymericnanoparticles. (Blank nanoparticles are particles which have no drugencapsulated, embedded, and/or adsorbed therein).

[0062] One advantage of particulate dosage forms of the activeingredient itself, or of polymeric particle dosage forms (e.g.nanoparticles), is the possibility of lyophilization and ofsterilization methods other than filter-sterilization. Thus, these formsof the active ingredient have an extended shelf-life and ease ofhandling.

[0063] In a preferred embodiment, the bisphosphonates may beencapsulated in nanoparticles (“NP”). Nanoparticles are 30-1000 nmdiameter, spherical or non-spherical polymeric particles. The drug canbe encapsulated in the nanoparticle, dispersed uniformly ornon-uniformly in the polymer matrix (monolithic), adsorbed on thesurface, or in combination of any of these forms. It is the submicronnature of this compositional form, which makes it more efficient intherapeutic applications. The submicron size facilitates uptake byphagocytic cells such as monocytes and macrophages, and avoids uptake inthe lungs. In a preferred embodiment, the polymer used for fabricatingnanoparticles is the biocompatible and biodegradable,poly(DL-lactide-co-glycolide) polymer (PLGA). However, any polymer whichis biocompatible and biodegradable may be used. Therefore, additionalpolymers which may be used to fabricate the NP include, but are notlimited to, polyanhydrides, polyalkyl-cyanoacrylates (such aspolyisobutylcyanoacrylate), polyetheyleneglycols, polyethyleneoxides andtheir derivatives, chitosan, albumin, gelatin and the like. The size ofthe nanoparticle used to encapsulate the active ingredient orbisphosphonate depends on the method of preparation and the mode ofadministration (e.g. IV, IA, etc.) Preferably, the nanoparticles rangein size from 70-500 nm. However, depending on preparation andsterilization techniques, the more preferred ranges include, but are notlimited to, 100-300 nm and 100-220 nm.

[0064] Encapsulating a small, hydrophilic, and charged drug such as thebisphosphonate in a nanoparticle is described herein. During thepreparation of the NP, there is a rapid diffusion of the drug into thewater phase, thus resulting in a low entrapment efficiency. Accordingly,a formulation was developed to overcome this low encapsulationefficiency. The following formulation parameters may influence drugentrapment efficiency and release properties: buffers, emulsifiers,stabilizers such as PVA, amount and molecular weight of PLGA polymers,type of BP, non-solvent type, timing, rate of mixing and evaporation ofthe ingredients, vacuum, and temperature. A cation, such as Ca⁺², anon-solvent, or other compounds may be incorporated with the BP, priorto encapsulation with the nanoparticle, in order to reduce thesolubility of the hydrophilic drug and increase its entrapmentefficiency. A “non-solvent” is immiscible with the polymer or drug butis miscible with the other solvent present and, as such, forces thepolymer or drug to leave its solvent. Several methods of nanoparticlepreparation known in the art may be used; the common methods including,but not limited to, emulsion or double-emulsion solvent-evaporationprecipitation methods, nanoprecipitation methods, coacervation methods,and solid-lipid liposomal methods.

[0065] Since the nanoparticles' surfaces are preferably negativelycharged due to the acidic functional groups of the polymer and/or theBP, increased uptake by phagocytic cells is expected, thereby leading toincreased activity against restenosis. Although particles which areneutral in charge may also be used to encapsulate the BPs, the mostefficient uptake by the monocytes/macrophages occurs with chargedparticles, with negatively charged particles being preferred.

[0066] The pharmaceutical carrier or diluent used in the composition ofthe invention may be any one of the conventional solid or liquid orsemisolid carriers known in the art. A solid carrier, for example, maybe lactose, sucrose, gelatins, and other carbohydrates. A liquidcarrier, for example, may be a biocompatible oil suitable for injectionsuch as peanut oil, water or mixtures of biocompatible liquids, or abiocompatible viscous carrier such as a polyethylene or gelatin gel.

[0067] The composition of the active ingredient used for injection maybe selected from emulsions, suspensions, colloidal solutions containingsuitable additives, and additional suitable compositions known to theskilled artisan.

[0068] The compositions of the invention may be administered by anyroute which effectively transports the active compound to theappropriate or desirable site of action. By a preferred embodiment ofthe invention, the modes of administration are intravenous (IV) andintra-arterial (IA) (particularly suitable for on-line administration).Other suitable modes of administration include intramuscular (IM),subcutaneous (SC), or intraperitonal (IP). Such administration may bebolus injections or infusions. The compositions may also be administeredlocally to the diseased site of the artery, for example, by means of amedical device which is coated with the active ingredient. Another modeof administration may be by perivascular delivery. Combinations of anyof the above routes of administration may also be used in accordancewith the invention.

[0069] The dosage of the active ingredient to be used also depends onthe specific activity of the active ingredient selected, on the mode ofadministration (e.g. systemic administration or local delivery), theform of the active ingredient (e.g. polymer, encapsulated in a particlesuch as a liposome, nanoparticle etc.), the size of the particle, andother factors as known per se.

[0070] In one embodiment, the dosage for alendronate in a PLGAnanoparticle preferably ranges from 0.015 mg/kg (per kg of body weight)to 3 mg/kg; more preferably, however, the dosage ranges from 0.15 to 1.5mg/kg. Dosages outside these preferred ranges may also be used, as canbe readily determined by the skilled artisan. When IV/IA injections orlocal delivery methods are used, i.e. via a balloon catheter, the dosageis at the lower end of the range. However, when IM or SC administrationmodes are used the dosage is approximately 10 times that used for IVadministration.

[0071] In accordance with a preferred embodiment of the invention,treatment of an individual with the active ingredient may be for thepurpose of preventing restenosis before its occurrence. For prevention,the active ingredient may be administered to the individual beforeangioplasty procedure, during the procedure or after the procedure aswell as combination of before, during and after proceduraladministration. Furthermore, the active ingredient may be administeredvia IV, IA, IM, SC, IP or any other suitable type of administration. Forexample, the active ingredient may be administered via IA the day of theangioplasty procedure (day 0), via IV the day before the procedure (−1)and/or on day 0, or both via IV the day before the procedure (−1) andalso after the procedural administration, for example, on day 6.

[0072] In accordance with a further embodiment of the invention, theactive ingredient is administered to an individual suffering fromrestenosis for the purpose of reducing or treating restenosis. In such acase, the active ingredient may also be administered to the individualat different periods of time after restenosis is discovered, eitheralone or in combination with other kinds of treatments.

[0073] In addition, the active ingredient may be administered before anyother conditions which may yield accelerated arteriosclerosis, as wellas acutely after the process has begun to inhibit further development ofthe condition.

EXAMPLES

[0074] The invention will now be demonstrated by way of non-limitingexamples with reference to the accompanying drawings. The animal modelsused in the examples below include the balloon-injured rat carotidarterial model and the balloon-injured hypercholesterolemic rabbitcarotid arterial model. The rat is an acceptable model in evaluating theantirestenotic effects of drugs and composites; however, the rabbit ismore preferred since it, unlike the rat, is both atherosclerotic andcontains a significant number of macrophages in the arterial wall.

Example 1

[0075] Liposomes of Clodronate

[0076] Stock solutions of clodronate were prepared by dissolving thedrug in deionized water at a concentration of 0.11 M, pH=7.

[0077] Liposome Preparation

[0078] 38.9 mg of distearoylphosphatidylglycerol (DSPG), 118.5 mg ofdistearoyl-phosphatidylcholine (DSPC) and 38.7 mg of cholesterol wereaccurately weighed and dissolved in 20 ml of chloroform: methanol (9:1)in a round bottom vial. The vial was gently warmed, and the solvent wasthen evaporated in rotavapor. 20 mls of hydrated diisopropylether werethen added and the vial was put into a water bath until the contentswere dissolved. 8 mls of the clodronate solution prepared as describedabove were then added, and the solution was sonicated at 55° C. for aperiod of 45 minutes. The organic phase was then evaporated in rotavapor(55° C., 100 rpm). Similarly, other drug-containing liposomes can beprepared.

[0079] Purification of Prepared Liposomes

[0080] A Sephadex gel was prepared by dissolving 2.6 grams of SephadexG-50 in 40 mls of water and stabilizing overnight. The column was rinsedwith 100 mls of buffer (50 mM Mes +50 mM HEPES +75 mM NaCl, pH 7.2). Theliposomes were applied to the column and the column was rinsed with thebuffer. The liposome was seen as a band which can be followed in thecolumn by its color. About 20 drops were collected from the column intoeach tube.

[0081] Animals

[0082] Animals were obtained and housed in the animal facilities of theFaculty of Medicine, The Hebrew University of Jerusalem, conforming tothe standards for care and use of laboratory animals of the HebrewUniversity of Jerusalem. Male rats of Sabra strain weighing 350-420 gwere used. The animals were fed standard laboratory chow and tap waterad libitum. All in vivo experiments were conducted under generalanaesthesia achieved with 80 mg/kg ketamine and 5 mg/kg xylazineadministered IP.

[0083] Rat Carotid Catheter Injury Model

[0084] The distal left common and external carotid arteries were exposedthrough a midline incision in the neck. The left common carotid arterywas denuded of endothelium by the intraluminal passage of a 2F ballooncatheter introduced through the external carotid artery. The catheterwas passed three times with the balloon distended sufficiently withsaline to generate a slight resistance. The catheter was then removedand the external carotid artery was ligated, and the wound was closedwith surgical staples.

[0085] Seven rats served as the control group, and 6 rats as the treatedgroup (randomly chosen). Liposomal clodronate was injected IV to the“treated group” one day prior to the arterial injury (6 mg of clodronateper rat) and repeated on day 6. In the control group similar injectionswere administered but with “empty” or blank liposomes (no clodronate).

[0086] All animals were sacrificed 14 days after injury by an overdoseof pentobarbital. Arteries were perfusion-fixed with 150 ml of 4%formaldehyde solution pH 7.4 at 100 mm Hg. The right atrium wasdissected and an 18G catheter connected to the perfusion system wasinserted in the left ventricle. The arterial segments were dissected,cut, gently separated from the polymer, and postfixed for at least 48hours in the same fixative solution. The arterial segments were embeddedin paraffin and cut at 8-10 sites 600 μm apart. Sections of 6 μm werethen mounted and stained with Verhoeff's elastin stain for histologicexamination.

[0087] Morphometric Analysis

[0088] The slides were examined microscopically by an investigatorblinded to the type of the experimental group. Six to eight sections ineach slide were evaluated by computerized morphometric analysis and theaveraged section data were further used as a representative of a wholeslide for comparisons between groups. The residual lumen, the areabounded by the internal elastic lamina (original lumen), and the areacircumscribed by the external elastic lamina (“total arterial area”)were measured directly. The degree of neointimal thickening wasexpressed as the ratio between the area of the neointimal and theoriginal lumen (% stenosis), and as the ratio between the neointimalarea to the area of the media (N/M). The medial area, an indirect indexof SMC viability, was determined as the difference between the totalarterial area and the original lumen area.

[0089] The surgical procedure and treatment did not cause mortality orapparent morbidity of the animals.

[0090] As seen in FIG. 1 the ratio between the neointimal area to thearea of the media (N/M) was significantly reduced following treatmentwith clodronate-encapsulated in liposomes. The N/M ratio in clodronatetreated rats was 0.28±0.23 as compared to 1.42±0.26 in the control group(mean±SD, p<0.01). Similarly as seen in FIG. 2, significant inhibitionof % stenosis was achieved in the treated group: 9.8±7.76 vs. 41.53±7.9,treated and control groups, respectively (mean±SD, p<0.01). There wereno apparent systemic side effects nor any effects on somatic growth asillustrated in FIG. 3.

[0091] Thus, the results of the experiments indicate that treatment ofrats with clodronate-containing liposomes significantly reducesrestenosis observed as neointimal formation following balloon-injury ofthe carotid artery.

Example 2

[0092] The antirestenotic effects of liposomal clodronate injectionswere studied in the balloon-injured rat and hypercholesterolemic rabbitcarotid arterial models. The rats were treated by clodronate-containingliposomes, empty liposomes (control), and clodronate in solution(additional control). The dose of clodronate injected was 1.5 and 15mg/kg administered one day before procedure (−1) and/or on day 6 (+6)post injury. The rabbits (following 30 days of atherosclerotic diet)were treated one day prior to balloon angioplasty by liposomalclodronate (10 mg/kg). The lumen, neointimal, medial and vessel areasand volumes were measured in the treated and control animal groups bydigital planimetry of histological sections, at 14 and 30 days postinjury in the rat and rabbit models, respectively.

[0093] The results of the antirestenotic effects of liposomal clodronateare shown in FIG. 4. As illustrated, no significant differences werefound between treatments with empty liposomes, and free clodronate insolution, which both exhibited marked neointimal formation. The extentof mean neointimal formation, mean neointimal to media ratio (N/M), and% stenosis following treatment with clodronate-laden liposomes wassignificantly reduced. However, the medial area was not affected by thevarious treatments indicating no deleterious effects on quiescent cells.Moreover, there were neither apparent systemic side effects nor anyeffects on bone and somatic growth. Significantly, more potenttreatments were evaluated, specifically, 1×15 mg/kg (−1) and 2×15 mg/kg(−1, and +6) injections, with no significant difference between them.Similar findings of no adverse effects were also observed in therabbits' study. Liposomal clodronate was significantly effective inreducing neointimal formation and % stenosis.

[0094] Furthermore, injection of silica particles also reduces intimalformation (FIG. 4). This observation can be attributed to the knowninhibiting effect of silica on macrophages.

[0095] The results of the experiment indicated that treatment byclodronate-containing liposomes significantly reduces neointimalformation following balloon-injury both in rat and rabbit models. Therewere neither apparent systemic and local side effects nor any effects onsomatic growth. It should be noted that although BPs are known asaffecting bone, no effects on the bone or on calcium and phosphoruslevels in bone and blood were observed following treatment withliposomal preparation of clodronate.

Example 3 Effect of ISA Composites on RAW 264 Proliferation

[0096] ISA Encapsulated in Nanoparticles

[0097] Nanoparticles (NP) were prepared by a novel solvent evaporationpolymer precipitation technique using a double emulsion system. 20 mg ofISA acid (Cohen, H. et al., 1999, Pharm. Res., 16: 1399-406) and 8.9 mgof NaHCO₃ were dissolved in 0.5 ml Tris buffer, and 90 mg of PLGA weredissolved in 3 ml dichloromethane. The aqueous sodium ISA solution wasadded to the PLGA organic solution and a water in oil (W/O) emulsion wasformed by sonication over an ice-bath using a probe type sonicator. ThisW/O emulsion was then added to a 2% polyvinyl alcohol (PVA) (20 ml)filter sterilized solution, and the pH was adjusted to 7.4 with NaOHsolution containing CaCl₂ in a molar ratio of 2:1 to ISA. The mixturewas mixed over an ice bath, forming a double emulsion (W/O/W). Theemulsion was stirred at 4° C. overnight to allow evaporation of theorganic solvent.

[0098] Nanoparticles which did not have any drugs encapsulated within(termed blank NP) were prepared according to the same procedure byomitting the drug. Ca⁺²-ISA salt with PVA and Ca+2-ISA salt wereprepared according to the same procedure by omitting the polymer or byomitting the polymer and the PVA, respectively. Ca⁺²-ISA was adsorbed onblank naoparticles (prepared as above) by dispersing the nanoparticlesin the buffer and precipitating ISA-calcium with the same ingredients asused above. The amount of drug entrapped in the NPs was determinedspectrophotometerically following sequential ultracentrifugation.

[0099] The influence of various formulation parameters on drugentrapment efficiency, release properties, and size have been examined.For example, the following parameters were evaluated: buffers,emulsifiers, various amounts of ISA, CaCl₂ (including without), amountof PVA (including without), different amounts and molecular weights ofPLGA/PLA polymers, temperature, yield and extrament efficiency. Thevarious formulation steps resulted in the development of sphericalnanoparticles containing ISA. Furthermore, NP formulationreproducibility was successfully demonstrated. High yield, entrapmentefficiency, as well as lyophilizability are important features for anynanoparticulate carrier.

[0100]FIG. 5 tabulates typical formulations of both ISA loadednanoparticles and blank nanoparticles. Whereas FIG. 5 illustrates atypical formulation, it shall be understood that additional formulationsmay also be effective. The formulation parameters tabulated include thesize of the nanoparticle both before and after lyophilization, thepercent recovery, the ISA entrapment (measuered as % of initial), thefinal ISA content, the ISA in NP, the ISA in the supernatant, theinitial ISA content, the Ca⁺² recovery, the Ca⁺² in the supernatant, theCa⁺² in NP, the ratio of Ca⁺² to ISA, and the Zeta potential. The sizeof the nanoparticle before lyophilization was 376 nm and increased 396nm after lyophilization. Typically, the size of the nanoparticles rangefrom 100-500 μm, depending not only upon preparation and sterilizationtechniques, but also upon the mode of administration. Lyophilization notonly increases the shelf-life of the nanoparticles, but also enablessteriliztion of the NP through irradiation. The percent recovery istabulated in FIG. 5 as 68.7%. Essential to the antirestenotic effect ofthe bisphosphonate NP is the bisphophonate content within thenanoparticle and the entrapment efficiency. These parameters aremeasured by: ISA entrampment, tabulated in FIG. 5 as 59.6%; final ISAcontent, measured as the ratio between ISA weight over the NP totalweight, and tabulated as 16.5%, but may range from 25 to 40%; the amountof ISA in NP, tabulated as 11.9 mg, but may be changed accordingly; theamount of ISA in the supernatant tabulated as 1.1 mg, but may range as afunction of the entrapment efficiency mentioned above; and the initialISA content, tabulated as 18.1%, but may be any percentage in anappropriate ratio to the polymer amount. In order to reduce thesolubility of the hydrophilic drug and increase its entrapmentefficiency, a cation, such as Ca⁺² is added to the composite. Theparameters associated with Ca⁺² include: Ca⁺² recovery, tabulated as72.3%; Ca⁺² in supernatant, tabulated as 38%; Ca⁺² in NP, tabulated as34%; and Ca⁺²/ISA, tabulated as 1.2 mols. However, these parameters mayrange as a function of the ratio between calcium to ISA, the type ofadditives, the pH of solution and other like factors. Additionally, theZeta potential is tabulated as −5.7. A negative value for thenanoparticle zeta potential is important for efficient uptake byphagocytic cells (e.g. macrophages).

[0101] Furthermore, lyophilized NPs were shown to have similarproperties to non-lyophilized NPs, in both in vitro and in vivoexperiments. Indeed, this is of significant importance since NPsterilization could be obtained through y irradiation of dry NP,ethylene-oxide sterilization, steam sterilization (when other polymersare used) or filter sterilization.

[0102] Each particle carrier (e.g., polymeric micro/nanoparticle)exhibited a certain entrapment efficiency for the bisphosphonate drug.The ISA entrapment efficiency in the NP reached 60% and is substantiallyhigher than any efficiency that is reported in the literature for anygiven hydrophilic drug in either PLGA nanoparticles or liposomes.

[0103] The bisphosphonate, ISA, serves as a model bisphosphonate. Thephysicochemical properties of other BPs are similar to those of ISA.Moreover, ISA serves as model drug for a low molecular weight,hydrophilic, charged molecule and, as such, was used as a modelbisphosphonate in the experiments herein. For properties of ISA, referto, Cohen H, Alferiev I S, Monkkonen J, Seibel M J, Pinto T, Ezra A,Solomon V, Stepensky D, Sagi H, Omoy A, Patlas N, Hagele G, Hoffman A,Breuer E, Golomb G, 1999, “Synthesis and preclinical pharmacology of2-(2-aminopyrimidinio) ethylidene-1,1-bisphosphonic acid betaine(ISA-13-1)-a novel bisphosphonate.” Pharm Res.;16:1399-406.

[0104] In Vitro Bioactivity

[0105] The effect of ISA on the growth of RAW 264 cells was determined.RAW 264 cells, which are derived from the murine macrophage cell lines,were plated at 2×10⁴ cells per well in 24-well plates and allowed togrow for approximately 24 hours in DMEM. The cells were then treatedwith various compositions of the ISA drug, specifically, free ISA, theISA-Ca² salt, and ISA encapsulated in a nanoparticle (“ISA NP”). Asdiscussed supra, the ISA NP contains Ca⁺² to lower the solubility of thebisphosphonate and increase its entrapment efficiency. For comparisonpurposes, the RAW 264 cells were also treated with blank NP, i.e.,nanoparticles with no drug embedded therein. The cells were thenanalyzed 48 hours after treatment. Analysis included cell counting byCoulter counter and cell viability by tryphan blue exclusion assay.

[0106] The effect of ISA compositions on the growth of RAW 264 cells invitro is illustrated in FIG. 6. The cell proliferation in buffer onlywas termed as 100%. The data represented is the mean with a ±SD(6≦N≦27). As the legend designates, *P<0.05, **P<0.01 in comparison tobuffer and $ P<0.01 in comparison to ISA+Ca² indicating that thedifferences are statistically significant.

[0107] As depicted in FIG. 6, free ISA had only a minor effect on thegrowth of RAW 264 cells (macrophages). However, the addition of equalCa⁺² concentrations to ISA to form a salt, potentiated thebishposphonates activity, and significantly suppressed the proliferationof RAW 264 cells in a dose response manner. Indeed, the use of ISA-Ca⁺²salt served as an appropriate control group to ISA NP activities sinceboth compositions contained equivalent molar amounts of Ca⁺² and ISA.ISA encapsulated in nanoparticles (“ISA NP”) were found to be potentinhibitors of the growth of RAW 264 cells. As illustrated in FIG. 6, theISA NP were far more potent than both ISA and ISA+Ca⁺². The blank NP hadno effect on the proliferation of RAW 264 cells up to 100 μM, indicatingthat the ISA NP growth inhibitory effect was caused by ISA and not bythe polymer.

[0108] Additionally, the effect of Ca⁺²-ISA salt formulations on RAW 264proliferation is presented in FIG. 7. The following formulations wereevaluated: Ca⁺²-ISA salt, Ca⁺²-ISA salt+PVA, and Ca⁺²-ISA adsorbed onthe surface of a blank NP. The cell proliferation in the buffer only wastermed as 100%. N.B., a lower column represents higher potency.

[0109] As depicted in FIG. 7, Ca⁺²-ISA salt potently inhibited theproliferation of RAW 264 cells at 50 μM, and the addition of PVA toCa⁺²-ISA salt further potentiated its activity. As discussed supra, PVAis an additive which is used to stabilize the BP-salt/complex. However,Ca⁺²-ISA adsorbed on the surface of the blank NP significantlysuppressed RAW 264 cell proliferation. As illustrated in FIGS. 6 and 7,the Ca⁺²-ISA salt (50 μM) adsorbed on a blank NP and ISA encapsulated inNP (50 μM) were comparable inhibitors of RAW 264 proliferation. Insummary, the results of the experiment indicate that Ca⁺²-ISA saltparticulates are useful in inhibiting restenosis by eliminating orinhibiting macrophages.

[0110] However, in vivo utilization of Ca⁺²-ISA salt particulates toeliminate macrophages, requires maintaining the composites in thenanometer size in order to be suitable for IV use. Maintaining the saltcomposites in the nanometer size may be achieved with a propersurfactant during the Ca⁺²-ISA salt preparation. Indeed, the benefit ofutilizing a Ca⁺²-ISA salt composite to treat restenosis is itssimplicity and the avoidance of PLGA use. However, a possible drawbackof this approach might be the rapid dissolution of the Ca⁺²-ISA saltseconds after administration, due to high dilution.

[0111] Similar experiments were conducted to determine the effect of ISANP on the proliferation of smooth muscle cells extracted from the aortasof adult male Sabra rats and 3T3 fibroblast cells. The results of theexperiment (not shown) indicate that ISA NP significantly inhibited thegrowth of rat SMC and 3T3 cells (fibroblasts).

[0112] In summary, ISA encapsulated within NP was found to inhibit thegrowth of the three main cell types involved in the restenotic cascade,namely macrophages (RAW 264 cells), fibroblasts (3T3 cells) andsmooth-muscle cells (SMC).

Example 4 Effect of ISA-Nanoparticles Rat Carotid Model of Restenosis

[0113] In Vivo Bioactivity

[0114] The following experiment examined the effect of ISA encapsulatedin NP, specifically PLGA based NP, on neointimal formation. The ISA NPwere prepared as described supra, in Example 3. Additionally, Male Sabrarats were used and prepared according to the rat carotid catheter injurymodel described supra, in Example 1. ISA NP was injected IV to the“treated group” one day prior to the arterial injury (−1 d) at a dosageof 15 mg/kg. In the control group, similar injections were administeredbut with blank NP, i.e. nanoparticles with no ISA encapsulated oradsorbed therein.

[0115] The animals were then sacrificed 14 days of injury, theirarterial segments dissected and prepared for histologic examination.(Refer supra, in Example 1, for details). The arterial segments wereevaluated by computerized morphometric analysis. The residual lumen, thearea bounded by the internal elastic lamina (original lumen), and thearea circumscribed by the external elastic lamina (total arterial area)were measured directly. The degree of neointimal thickening wasexpressed as the ratio between the area of the neointimal and theoriginal lumen (% stenosis), and as the ratio between the neointimalarea to the area of the media (N/M). The medial area, an indirect indexof SMC viability, was determined as the difference between the totalarterial area and the original lumen area.

[0116] The experimental results indicate that administration of ISA NPsignificantly inhibited vascular neointimal formation in comparison toblank NP treatment. As indicated in FIG. 8A, significant inhibition of %stenosis was achieved in the ISA NP treated group (n=12). Similarly, asillustrated in FIG. 8B, the extent of mean neointimal formation and meanneointimal to media ratio (N/M) following treatment with ISA NP wassignificantly reduced. Thus, the experimental results indicate thattreatment of rats with ISA encapuslated nanoparticles via IVadministration significantly reduces restenosis observed as neointimalformation following balloon-injury of the carotid artery.

[0117] Additionally, SC administration of ISA encapsulated innanoparticles was evaluated and found to significantly inhibitneointimal formation 14 days after vascular injury. However, the SCadministration provided a weaker restenosis inhibiting effect than thatobtained from IV delivery of the ISA NP.

[0118] In summary, the above experiments indicate that the improvedstability, high drug entrapment efficiency, and increased bioactivity ofISA encapsulated in NP or ISA absorbed on NP, possess novel andimportant advantages for clinical applications. Additionally, Ca-BP saltparticulates were also found to be bioactive and effective in inhibitingproliferation of monocytes.

Example 5 Effect of Alendronate-Nanoparticles in Rabbit Model ofRestenosis

[0119] Alendronate NPs

[0120] Alendronate encapsulated within nanoparticles (“alendronate NPs”)were prepared by a novel solvent evaporation polymer precipitationtechnique using a double emulsion system. 20 mg of alendronate weredissolved in 0.5 ml Tris buffer with 2.8% PVA and 90 mg of PLGA weredissolved in 3 ml dichloromethane. The aqueous alendronate solution wasadded to PLGA organic solution and a water in oil (W/O) emulsion wasformed by sonication over an ice bath using a probe type sonicator, at14 W for 90 seconds. This W/O emulsion was further added to 10.5 ml ofTris buffer (containing 2% PVA and CaCl₂ solution in molar ratio 2:1 toalendronate), and sonicated for 90 seconds over an ice bath, forming thedouble emulsion (W/O/W). The emulsion was stirred at 4° C. for 3 hours,to allow evaporation of the organic solvent.

[0121]FIG. 9 tabulates the formulation parameters for the alendronate NPformed. Although FIG. 9 tabulates a typical alendronate NP, it shall beunderstood that various formulations may also be effective. Theformulation parameters include size, alendronate entrapment, the amountof alendronate in NP, the alendronate in supernatant, the initial amountof alendronate, the PLGA amount and the volume of a 0.246M calciumchloride solution. The size of the nanoparticle was 219 nm. As discussedsupra, typically, the size of the nanoparticles range from 100-500 nm,depending not only upon preparation and sterilization techniques, butalso upon the mode of administration. Essential to the antirestenoticeffect of the bisphosphonate NP is the bisphophonate content within thenanoparticle and the entrapment efficiency. These parameters aremeasured by: alendronate entrapment, tabulated in FIG. 9 as 55.1%;alendronate in NP, tabulated as 1.002 mg/ml; alendronate in thesupernatant, tabulated as 0.232 mg/ml; the initial amount ofalendronate, tabulated as 20 mg; the PLGA amount, tabulated as 90 mg;and the volume of calcium chloride, tabulated as 0.5 ml. However, theseparameters may be modified to provide additional formulations. In theexperiment, the effect of alendronate NP on RAW 264 proliferation wasevaluated. For procedures describing the growth of RAW 264 cells andsubsequent treatment with NP refer to Example 3, supra. As depicted inFIG. 10, alendronate NP are potent inhibitors of macrophages, whoseactivity increases with concentration.

[0122] Balloon-Injured Hypercholesterolemic Rabbit Arterial Model

[0123] The antirestenotic effect of alendronate NPs was evaluated inboth the balloon-injured rat and the balloon-injuredhypercholesterolemic rabbit carotid arterial models. While alendronateNPs were successful in reducing restenosis in both models, ofsignificant importance is the marked efficacy discovered in thehypercholesterolemic model of balloon-injured rabbits. The rabbits weretreated by alendronate NPs and blank NPs (control) via SC and IVadministration in order to compare the differences between the two modesof administration. A group of rabbits were treated one day prior toballoon angioplasty with 1.5 mg/kg of alendronate NPs via SCadministration. For comparison purposes, two additional groups were alsotreated one day before the procedure (-Id) with 0.15 mg/kg ofalendronate NPs via SC and IV administration. The lumen, neointimal,medial and vessel areas were measured in the treated and control rabbitgroups by digital planimetry of histological sections at 30 days postinjury.

[0124] The results of the antirestenotic effects of alendronate NPs inthe balloon-injured hypercholesterolemic rabbit model are illustrated inFIGS. 11, 12a and 12 b.

[0125]FIG. 11 illustrates that the % restenosis following treatment withalendronate NPs via SC administration was significantly reduced.

[0126]FIGS. 12a and 12 b compare the effect of two dosage amounts of thealendronate NPs via SC administration, specifically 1.5 and 0.15 mg/kgand also the effects of two modes of administration, IV and SC. Asillustrated in the figures, the mean neointimal to media ratio (N/M) and% stenosis was reduced in a dose response manner. Specifically, a doseof 1.5 mg/kg was more effective in reducing N/M and % restenosis incomparison to 0.15 mg/kg. Moreover, the inhibition of neointimalformation and % restenosis by alendronate NPs via SC administration wasslightly greater than that obtained via IV delivery, although notsignificant in light of the experimental standard deviation.Additionally, there were neither apparent systemic side effects nor anyeffects on bone and somatic growth.

[0127] In conclusion, alendronate NP is a highly potent inhibitor ofrestenosis in animal models via SC and IV administration. Furthermore,the dosage range of 0.15 to 1.5 mg/kg was found to be the most potentdelivery system in preventing restenosis.

Example 6 Effect of Alendronate-Nanoparticles in Human Blood

[0128] In this example, the ability of alendronate NPs to decrease thenumber of monocytes in human blood was studied. Human blood was drawn toEDTA-containing test tubes and 200 μl were incubated for 24h in 37° C.on a shaker with the indicated doses of alendronate-nanoparticles in 50μl diluted in 50 of PBS. Control samples were incubated with 50 μl. Thesamples were then incubated (30 min. 4° C., in the dark) withRPE-conjugated anti-CD14 Ab (specific for monocytes) for 30 min. RBC(red blood cells) were lysed by FACS lysing solution (Becton-Dickinson,San-Jose, Calif.) and distilled water, and following washings in FACS(fluorescence activated cell sorting) solution, flow cytometry analysiswas performed. Monocytes were detected by side-scattering andfluorescence.

[0129] The results of the anti-proliferative effects ofalendronate-nanoparticles on monocytes in human blood are illustrated inFIG. 13. It is apparent that alendronate NPs potently decreased theamount of monocytes in human blood in a dose response manner. Sincemonocytes, in their normal state, are recruited to the areas traumatizedby angioplasty or other intrusive intervention and initiate theproliferation of smooth-muscle cells, thus leading to restenosis,inhibiting the number of monocytes will inhibit restenosis.

We Claim:
 1. A method of treating or preventing restenosis, comprisingadministering to an individual an effective amount of an activeingredient selected from the group consisting of a bisphosphonate, abisphosphonate salt, a bisphosphonate ester, and a bisphosphonatecomplex, wherein the active ingredient is in a particle dosage form,thereby treating or preventing restenosis.
 2. A method of treating orpreventing restenosis, comprising administering to an individual aneffective amount of an active ingredient selected from the groupconsisting of a bisphosphonate, an insoluble bisphosphonate salt, aninsoluble bisphosphonate ester, and an insoluble bisphosphonate complex,wherein the active ingredient is in a free particulate dosage form,thereby treating or preventing restenosis.
 3. The method according toclaim 1 or 2, wherein the particle or particulate is of a size taken-upby target cells of the white blood-cell lineage and other phagocyticcells.
 4. The method according to claim 3, wherein the target cells areselected from the group consisting of monocytes and macrophages.
 5. Themethod according to claim 1, wherein particles are selected from thegroup consisting of polymeric particles, liposomes, microparticles,nanoparticles, microspheres, and nanospheres.
 6. The method according toclaim 2, wherein free particulates are selected from the groupconsisting of aggregates, flocculates, colloids, polymer chains,insoluble salts and insoluble complexes.
 7. The method according toclaim 1, wherein the active ingredient is encapsulated within theparticle.
 8. The method according to claim 1, wherein the activeingredient is embedded within the particle.
 9. The method according toclaim 1, wherein the active ingredient is adsorbed on the particlesurface.
 10. The method according to claim 1 or 2, comprisingadministering to the individual an active ingredient, having thefollowing formula (I):

wherein R₁ is H, OH or a halogen atom; and R₂ is a halogen; linear orbranched C₁-C₁₀ alkyl or C₂-C₁₀ alkenyl optionally substituted byheteroaryl or heterocyclyl C₁-C₁₀ alkylamino or C₃-C₈ cycloalkylaminowhere the amino may be a primary, secondary or tertiary; —NHY where Y ishydrogen, C₃-C₈ cycloalkyl, aryl or heteroaryl; or R₂ is -SZ where Z ischlorosubstituted phenyl or pyridinyl.
 11. The method according to claim1 or 2, wherein said active ingredient is clodronate, etidronate,tiludronate, pamidronate, alendronate, risendronate or ISA.
 12. Themethod according to claim 1 or 2, wherein the administration isintravenous (IV), intrarterial (IA), intramuscular (IM), subcutaneous(SC), intraperitoneal (IP), or delivered by a ‘sweating balloon’, acoated balloon or on a coated stent.
 13. The method according to claim 1or 2, wherein the active ingredient is administered before anangioplasty procedure.
 14. The method according to claim 1 or 2, whereinthe active ingredient is administered the day of an angioplastyprocedure.
 15. The method according to claim 1 or 2, wherein the activeingredient is administered after an angioplasty procedure.
 16. A methodof treating or preventing restenosis, comprising administering to anindividual an effective amount of a compound, wherein said compoundinhibits blood monocytes or tissue macrophages, thereby treating orpreventing restenosis.
 17. The method according to claim 16, wherein thecompound is selected from the group consisting of gallium and gold. 18.A method of treating or preventing restenosis, comprising administeringto an individual an effective amount of a bisphosphonate nanoparticle,thereby treating or preventing restenosis.
 19. A pharmaceuticalcomposition comprising an active ingredient selected from the groupconsisting of a bisphosphonate particle, a bisphosphonate particulate, asalt, an ester and a complex of bisphosphonate, together with apharmaceutically acceptable carrier for the prevention or treatment ofvascular restenosis.
 20. The pharmaceutical composition according toclaim 19, wherein the active ingredient is together with a diluent. 21.The pharmaceutical composition according to claim 19, wherein the activeingredient is together with a stabilizer.
 22. The pharmaceuticalcomposition according to claim 19, wherein the bisphosphonate isencapsulated in the particle.
 23. The pharmaceutical compositionaccording to claim 19, wherein the bisphosphonate is embedded in theparticle.
 24. The pharmaceutical composition according to claim 19,wherein the bisphosphonate is adsorbed on the surface of the particle.25. The pharmaceutical composition according to claim 19, wherein theparticle is of a size taken-up by target cells of the white blood-celllineage.
 26. The pharmaceutical composition according to claim 25,wherein the target cells are selected from the group consisting ofmonocytes and macrophages.
 27. The pharmaceutical composition accordingto claim 19, wherein the particles are selected from the groupconsisting of polymeric particles, liposomes, microparticles,nanoparticles, microspheres, and nanospheres.
 28. The pharmaceuticalcomposition according to claim 19, wherein the free particulates areselected from the group consisting of aggregates, flocculates, colloids,polymer chains, insoluble salts and insoluble complexes.
 29. Thepharmaceutical composition according to claim 19, wherein thebisphosphonate has the following formula (I):

wherein R₁ is H, OH or a halogen atom; and R₂ is a halogen; linear orbranched C₁-C₁₀ alkyl or C₂-C₁₀ alkenyl optionally substituted byheteroaryl or heterocyclyl C₁-C₁₀ alkylamino or C₃-C₈ cycloalkylaminowhere the amino may be a primary, secondary or tertiary; —NHY where Y ishydrogen, C₃-C₈ cycloalkyl, aryl or heteroaryl; or R₂ is —SZ where Z ischlorosubstituted phenyl or pyridinyl.
 30. The pharmaceuticalcomposition according to claim 19, wherein said active ingredient isclodronate, etidronate, tiludronate, pamidronate, alendronate, or ISA.31. The pharmaceutical composition according to claim 19, forintravenous (IV), intrarterial (IA), intramuscular (IM), subcutaneous(SC), or intraperitoneal (IP) administration.
 32. The pharmaceuticalcomposition according to claim 19, wherein the active ingredient isadministered before an angioplasty procedure.
 33. The pharmaceuticalcomposition according to claim 19, wherein the active ingredient isadministered the day of an angioplasty procedure
 34. The pharmaceuticalcomposition according to claim 19, wherein the active ingredient isadministered after an angioplasty procedure.